The goal of this project is to understand how the C-terminal domain of RNA polymerase II is used to couple transcription with several post-initiation steps in gene expression. These events include mRNA capping, splicing, and polyadenylation, as well as regulation of transcription elongation and termination. Current data supports a model in which the pattern of GTD phosphorylation changes at different stages of transcription. Each phosphorylation state may be recognized by a distinct set of CTD-interacting proteins. This allows a dynamic exchange of elongation and mRNA processing factors, each one recruited at the appropriate time(s) of the transcription cycle. The experiments in this project will test this model and identify physical and functional relationships between the CTD, its various kinases and phosphatases, and associated elongation and mRNA processing factors. Four specific aims are proposed. In the first aim, we will explore the decision between two pathways for transcription termination: the mRNA polyadenylation/torpedo mechanism and the snoRNA pathway. The role of protein factors, RNA sequences, and CTD phosphorylation will be probed. The second aim will continue our chromatin immunoprecipitation studies of the crosslinking patterns of elongation and mRNA processing factors in various mutant strains. Changes in crosslinking patterns of one factor when a second is mutated suggests an interdependence that will be tested biochemically and genetically. Specific Aim 3 will be to continue our work on developing in vitro systems for reproducing the CTD modification and processing events observed in vivo. These in vitro systems will be used to test and extend the models of factor interactions derived from Aims 1 and 2. Specific Aim 4 will be to better characterize the patterns of CTD phosphorylation using mass spectroscopic techniques. The experiments proposed will significantly extend our understanding of how various steps in gene expression are integrated. It is clear that post-initiation events are regulated in many systems for modulation of gene activity. A clear understanding of the fundamental mechanisms of gene expression will provide the groundwork for future therapies, including gene replacement therapies and direct modulation of cellular and viral gene expression.